Advanced stage colorectal cancer is often unsuccessfully treated using standard therapies, and many novel immune-based therapies are under development. Despite major advances in the field of tumor immunology, and detectable anti-tumor immune responses, most approved and investigational cancer immunotherapies (CIT) have shown only marginal clinical tumor regressions. This outcome is thought to be the result of a varie- ty of immunosuppressive mechanisms induced within tumors, such as the induction of CD4+ regulatory T cells (TREGS). TREGS are capable of directly inhibiting anti-tumor attack by immune cells, resulting in tumor escape from immune elimination. TREGS have been shown to infiltrate many human cancers, including colorectal can- cer, and suppress immune attack. Identifying therapies that could reverse induction of TREGS is essential to im- proving immunotherapy strategies. Recent studies have revealed that sub-lethal doses of ionizing radiation (IR) have immuno-modulatory effects on tumor cells, both in vitro and in vivo. This property of IR can be ex- ploited when used in combination with immune-based cancer treatments. For these combination strategies to be most effective investigators need to determine exactly how IR can be optimized to specifically serve CIT. At present, the extent of immune relevant changes induced by IR is unclear. It is also unclear which cellular im- mune responses can be successfully modified following tumor irradiation. One intriguing possibility is that in- creased immune responses following IR could occur through inhibition or reduction of TREGS, thus allowing anti- tumor immune cells to function. The hypothesis explored here is that exposure of human colorectal carcinoma cells to sub-lethal doses of IR will induce a molecular signature of enhanced immunogenicity that results in de- creased TREG activity. The goal of this study is to define the impact of tumor irradiation on TREGS. Work pro- posed here will evaluate colorectal carcinoma cells to determine if they each respond similarly to IR. The im- pact of tumor irradiation on TREG number and function will also be assessed. The specific aims include: 1) to qualify and quantify changes in the expression of molecules involved in regulating TREGS in irradiated human tumor cells, 2) to evaluate the ability of irradiated tumor cells to reduce TREG cell numbers, and 3) to evaluate the ability of irradiated tumor cells to reduce TREG cell function. Colorectal carcinoma cells will be evaluated for changes in the expression of genes implicated in TREG recruitment, activation, induction and inhibition. Exper- iments will evaluate the impact of irradiated colorectal carcinoma cells on TREG cell proliferation, viability and function. Results of the proposed research will expand information available regarding immune relevant mo- lecular changes induced in carcinoma cells following IR. This award will facilitate progress towards achieving the long-term goal of elucidating the mechanistic link between IR and increased tumor attack by immune cells. Collectively, we expect the outcomes of this innovative study to lead to novel combinations of immune thera- pies with IR for the control and treatment of cancer. PUBLIC HEALTH RELEVANCE: 1 in 20 persons will be diagnosed with cancer of the colon and rectum during their lifetime, and advanced stage disease continues to be unsuccessfully treated. The work proposed here will evaluate colo- rectal carcinoma cells to determine how they may benefit from combination radiation/immunotherapy on a cel- lular level. A major goal of this study is to define ways to maximize synergy when combining radiation with immune-based strategies, and data obtained from these studies could be translated into the clinic for the ra- tional design of combination radiation/immunotherapies for the control and treatment of colorectal cancer.